Hair graying is the most noticeable and least innocuous of aging phenotypes. Thus, on two fronts, hair graying lends itself to the comprehensive study of age-related tissue decline because it is not lethal and because of its outwardly visible phenotype. We know that gray hair is caused by the depletion of melanocyte stem cells in the hair follicle, but how aging influences melanocyte stem cell maintenance biologically remains largely unknown. Previously, we demonstrated that regulation of the transcription factor Sox10 is essential for McSC maintenance. McSCs require Sox10 for their survival, but overexpression of Sox10 drives aberrant McSC differentiation leading to McSC loss and eventual hair graying. Interestingly, nearly all other hair graying models, including age-related graying in humans, exhibit the same McSC differentiation phenotype. The fact that SOX10 is a critical factor in melanocyte lineage progression lead me to hypothesize that McSC identity is dependent on the proper integration of upstream signaling pathways and the regulation of melanocyte differentiation via Sox10. To test this I use a genome-wide approach in Aim 1, to first evaluate the contribution of stem cell genes and lineage specific genes in the driving McSC identity. I then define the relative importance of regulating lineage-specific transcription factors, namely SOX10, in maintaining this identity. In Aim 2, I combine cell biological and genome-wide analysis to understanding the etiology of hair graying with age and address the applicability of studying McSC maintenance as a bonafide age-related phenotype. In this aim I extend my comparisons to aged hair follicle stem cells to elucidate commonalities in aged stem cells that reside in the same niche. Aim 3 provides a realistic example of how the McSC model can be used to test specific mechanisms that participate in McSC maintenance. These studies serve as the basis for using this model in future research and screens looking for novel genes and pathways involved in tissue homeostasis, and those that are particularly affected with age. The research plan for this K99/R00 application describes my investigation into the biological pathways involved in maintaining adult stem cells over time. In particular, I a interested in exploiting the melanocyte stem cells and hair graying model system to identify new genes and proteins that may participate in the aging process. My approach is to use in-vivo mouse models in combination with modern cell biology techniques (FAC sorting) and whole genome analysis (RNAseq) to study melanocyte stem cells and hair graying. My training background in genetics, cell biology and developmental biology makes me particularly suitable for this project as I have experience in mouse transgenics and the cellular assays needed for quantitative, in-vivo phenotypic analysis. I view my K99 training as a segue-way into the aging field and an opportunity to bring a more functional genetics perspective to the hair graying model. Thus I have set up a unique mentoring team to guide me in this effort including Dr. Bill Pavan, a genetic and genomics expert, Dr. Toren Finkel, a aging and cellular metabolism expert, and Dr. Tom Hornyak, an expert in melanoma, pigment cell and stem cell biology. The ability to perform research at the National Human Genome Research Institute of NIH, provides me unique opportunities to train with experts in state-of-the-art DNA/RNA sequencing technologies and bioinformatics. During the K99 portion of application I have outlined specific career training that includes both hands-on experience (in lab mentoring of students and co-teaching a graduate course) and lecture-based learning (training courses in management and how to start a lab). In the short term, my goals are to further dissect the molecular regulation of melanocyte stem cells and hair graying while also gaining the tools needed to excel in a science and teaching career. I also intend the K99 to lead to new collaborations that will serve me well in the R00 phase and give me perspective on the fields of aging, genomics, and stem cell biology. Together these two aspects of the K99 will result in a research and mentoring/teaching platform that will support the successful implementation and completion of the R00 phase of this award. In the long term, I intend to build a career in biological aging studies, using this hair graying model to investigate cellular pathways that are important in maintaining stem cells over time, and make an opportunity for myself in training future scientists interested in this field.